Carlos Orestes Martin Medina

• Dixit, Pooja; Benavente, Verónica; Gustafsson, Tomas; Hedenström, Mattias; Gorzsás, András & Sundman, Ola [Show all 9 contributors for this article] (2025). Process development and pilot-scale validation of γ-valerolactone pretreatment for softwood biorefining. Biomass & Bioenergy. ISSN 0961-9534. 208. doi: 10.1016/j.biombioe.2025.108846. Full text in Research Archive Show summary

  Developing effective fractionation methods remains challenging in biorefining. γ-Valerolactone (GVL) is a promising green solvent, yet its application in softwood biorefineries is still underexplored. In this study, GVL pretreatment for softwood biorefining was assessed at laboratory and pilot scales. The effects of temperature (170–210 °C), time (20–60 min), and GVL-to-water ratios (20:80 – 80:20 %) on the biorefining of spruce sawdust were initially investigated through lab-scale experiments using a 1-L reactor. A GVL/water solution at a 40:60 ratio, assisted by 0.4 g of sulfuric acid per 100 g of biomass, enabled the solubilization of up to 81.4 % of lignin and nearly the entire hemicellulosic fraction, while effectively preserving cellulose, which was subsequently saccharified with over 90 % conversion. Lignin was regenerated from the liquors and characterized using pyrolysis-gas chromatography/mass spectrometry, high-performance size-exclusion chromatography, Fourier-transform infrared spectroscopy, and 1H-13C heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy. The biorefinery concept was successfully scaled up and validated at pilot scale in a 50-L reactor, where enzymatic saccharification of the resulting cellulosic pulp produced hydrolysates that, upon fermentation, yielded 231.4 g of ethanol per kilogram of pulp. Lignin regenerated from the pilot-scale pretreatment liquors (118.9 g per kilogram of raw sawdust) and the lignin-rich saccharification residue (182.7 g/kg) were subjected to hydrothermal liquefaction, and the resulting biocrudes were characterized to assess their potential for biofuel formulation. The study showed the suitability of GVL for spruce biorefining to achieve high recovery of digestible cellulose, lignin and hemicelluloses fractions, that are subsequently valuable for chemicals and fuels production.

• Momayez, Forough; Okoro, Oseweuba Valentine; Shavandi, Armin; Martín, Carlos; Denayer, Joeri F.M. & Karimi, Keikhosro (2025). A critical review of ultrasonication as a green technology for enhanced biomass valorization in bioethanol and biogas production. Process Safety and Environmental Protection (PSEP). ISSN 0957-5820. 200. doi: 10.1016/j.psep.2025.107334. Full text in Research Archive Show summary

  This paper critically reviews and discusses the utilization of ultrasound as an eco-friendly approach for enhancing biomass valorization efficiency. In contrast to other methods, this process requires no additional chemicals, no post-treatment (such as wastewater treatment), and helps achieve industrial electrification goals. Ultrasound irradiation has been employed as a powerful tool in biomass pretreatment and biorefining, as well as a green extraction method for recovering bioactive molecules. By partially degrading lignin structures, ultrasound irradiation significantly improves the biological conversion of waste materials and lignocellulose, facilitating the exposure of valuable macromolecules, i.e., cellulose and hemicelluloses. This research begins with an introduction to sonication technology, and subsequently presents comprehensive discussions focusing on the application of ultrasound in the pretreatment of sludge and lignocellulosic materials for anaerobic digestion. Furthermore, various facets of ultrasound usage in bioethanol production, including substrate pretreatment, enzymatic and acid hydrolyzes, and fermentation techniques, are also examined. Additionally, the benefits of employing ultrasound technology to recover high-value, heat-sensitive bioactive compounds at temperatures below their degrading points, thereby preserving their functionality, are explored. Looking ahead, this review explores current trends in ultrasound technology adoption and its potential for scaling up and commercialization, introducing pathways for a more sustainable and efficient approach for biomass valorization.

• Miranda, Diego A.; Carrasco, Cristhian; Soto, Luis Alejandro Romero; Lundqvist, Jenny; Sundman, Ola & Hedenström, Mattias [Show all 10 contributors for this article] (2025). Sustainable production of exopolysaccharides from quinoa stalk hydrolysates using halotolerant Bacillus swezeyi: fermentation kinetics and product characterization. Biofuels, Bioproducts and Biorefining. ISSN 1932-104X. 19(5), p. 1326–1348. doi: 10.1002/bbb.70021. Full text in Research Archive Show summary

  Abstract Microbial exopolysaccharides (EPSs) have attracted increasing attention due to their versatile applications across diverse areas. However, large‐scale production of EPSs remains challenging due to the high production costs, primarily driven by the use of synthetic carbon sources. This study demonstrates the potential of quinoa stalk hydrolysates as a sustainable alternative for EPS production using a halotolerant bacterial strain that was isolated from a hypersaline environment and termed SU4M. The bacterial isolate was identified through 16S rRNA and gyrB sequencing as a Bacillus swezeyi strain, and was then cultivated in quinoa stalk hydrolysates. The hydrolysates were produced by acid‐catalyzed hydrothermal pretreatment using either sulfuric acid or phosphoric acid, followed by enzymatic saccharification. Fermentation experiments conducted in both shake flasks and bioreactors demonstrated that B. swezeyi SU4M utilized glucose from the hydrolysates efficiently, resulting in significantly higher biomass (5.1 ± 0.1 g L −1 ) and EPS production (1.2 ± <0.1 g L −1 ) compared to synthetic media (4.3 ± 0.1 g L −1 and 1.1 ± <0.1 g L −1 ). The kinetic analysis revealed distinct substrate consumption rates and growth patterns, with hydrolysates enhancing EPS yields under single‐pulse fed‐batch conditions. Advanced characterization techniques, including compositional analysis, Fourier transform infrared (FTIR) spectroscopy, 1 H and 1 H‐ 13 C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR), high‐performance size‐exclusion chromatography (HPSEC), and thermogravimetric analysis (TGA), confirmed that the EPSs derived from hydrolysates were heteropolysaccharides with close structural similarities to those obtained from synthetic media. These findings underscore the potential of quinoa stalk hydrolysates as a biobased alternative to synthetic media as a substrate for EPS production.

• Tang, Chaojun; Martín, Carlos & Jönsson, Leif J. (2025). Effects of aeration of softwood pretreatment liquid on inhibitors and fermentability using Saccharomyces cerevisiae yeast. Biotechnology for Biofuels and Bioproducts. 18. doi: 10.1186/s13068-025-02708-4. Full text in Research Archive Show summary

  Background Aeration plays a critical role in the bioconversion of pretreated lignocellulose by enhancing lytic-polysaccharide-monooxygenase(LPMO)-supported enzymatic saccharification. However, its broader impact, particularly on fermentation inhibitors, remains insufficiently understood. The hypothesis that aeration not only promotes LPMO activity, which has been shown clearly in previous studies, but also affects fermentation inhibitors was investigated in experiments with softwood pretreatment liquids. The effects of aeration were explored through chemical analysis of fermentation inhibitors and through subsequent fermentations with the xylose-utilizing Saccharomyces cerevisiae yeast CelluX™4 to test the fermentability. Controls in which N 2 rather than air was supplied to the pretreatment liquids were used to distinguish between evaporation effects and effects caused by oxidation due to O 2 in air. In separate experiments, two redox-dependent detoxification methods, treatments with sulfite and laccase, were further investigated. Results While aeration had no negative effects on the subsequent fermentation of a sugar control, it compromised the fermentability of the pretreatment liquids. Compared to the N 2 control, subsequent fermentation of aerated samples showed reduced consumption of fermentable sugar (glucose, mannose, xylose) at 0.61 compared to 0.76 g L −1  h −1 , and lower ethanol productivity (0.23 vs. 0.30 g L −1  h −1 ). Apart from more commonly studied pretreatment by-products (such as aliphatic carboxylic acids, furan aldehydes, and phenolics), methanol (~ 1 g L −1 ) was detected in both pretreatment liquids. The methanol concentration decreased during gas addition, which was attributed to evaporation. Compared to the initial pretreatment liquid, aerated reaction mixtures exhibited slightly elevated levels of formaldehyde, but lower levels of furfural and vanillin. Sulfite detoxification was successful under both aeration and N 2 conditions. Treatment with laccase was found to have variable effects on the fermentability depending on the conditions applied. Conclusions The results underscore the dual role of aeration in softwood bioconversion, positive for promoting LPMO activity but potentially negative with respect to subsequent fermentability, and highlight the need to carefully tailor aeration strategies for the design of efficient biochemical processing of lignocellulosic feedstocks. Treatment with reducing agents, such as sulfite, emerges as a possibility to alleviate negative side-effects on the fermentability when aeration is used to promote LPMO activity.

• Miranda, Diego A.; Fernández, Lucía; Strætkvern, Knut Olav; Carrasco, Cristhian; Martín, Carlos & Soto, Luis Alejandro Romero (2025). Production of exopolysaccharides by halotolerant bacteria cultivated in hydrolysates of spent mushroom substrate and hydrothermally pretreated Jerusalem artichoke stalks. Industrial crops and products (Print). ISSN 0926-6690. 236. doi: 10.1016/j.indcrop.2025.121961. Full text in Research Archive Show summary

  Global issues over the unsustainable use of resources demand novel bio-based processes. The production of edible mushrooms from forest and agricultural residues offers efficient bio-based solutions. Spent mushroom substrate (SMS), a by-product from mushroom cultivation, can be valorized as a source of fermentable sugars. Similarly, the stalks of Jerusalem artichoke (Helianthus tuberosus), a crop of increased interest, represent under-investigated bioresources to be considered for biorefinery applications. In this study, SMS and Jerusalem artichoke stalks (JAS) were used for the first time as substrates to produce exopolysaccharides (EPS) by cultivating halotolerant bacteria (coded SU4M and SU3A) isolated from a hypersaline environment. Cultivations were performed using either cellulosic hydrolysates obtained by enzymatic saccharification or synthetic media. Acid-catalyzed hydrothermal pretreatment was applied to enhance the enzymatic saccharification of JAS, while no pretreatment was needed to saccharify SMS. The use of a SMS hydrolysate-based medium resulted in the highest biomass concentration (3.8 g L−1) and EPS production (852.1 mg L−1) among the media used in shake-flask fermentations. The fermentations in SMS hydrolysates resulted in comparable EPS volumetric productivity (17.8 ± 0.7 mg L−1 h−1) to that of synthetic medium (16.8 ± 0.8 mg L−1 h−1), but higher than that of the JAS hydrolysates (13.3 ± 0.6 mg L−1 h−1). Cultivation in SMS hydrolysates was particularly advantageous, as it did not require either pretreatment or external nutrient supplementation. The highest volumetric productivity of EPS production was achieved in a bioreactor when SMS hydrolysate was used, reaching 100 mg L⁻¹ h⁻¹ . This study demonstrates the potential of spent mushroom substrate and Jerusalem artichoke stalks, two underexploited agroresidues, as sustainable substrates for EPS production, thereby contributing to the strengthening of the regional bioeconomy in rural areas.

• Martín, Carlos; Xiong, Shaojun & Zervakis, Georgios I. (2025). Exploiting the Biorefinery Potential of Spent Mushroom Substrate: The Time to Do It Is Now. Molecules. ISSN 1431-5157. 30(23). doi: 10.3390/molecules30234518. Full text in Research Archive Show summary

  Spent mushroom substrate (SMS), the residual material left after mushroom cultivation, represents an abundant yet underutilized bioresource. With global mushroom production generating millions of tons of SMS annually, its disposal constitutes a missed opportunity within the circular bioeconomy. This Opinion article highlights why SMS should be repositioned as a valuable raw material for sustainable biorefineries and outlines the technological, economic, and regulatory steps needed to unlock its potential.

• Qi, Shengxu; Martín, Carlos; Xiong, Shaojun; Xie, Jiale & Chen, Feng (2025). Fungal pretreatment of hardwood for cellulosic ethanol production: Formation of by-products and the potential effects on downstream bioconversion processes. Industrial crops and products (Print). ISSN 0926-6690. 237. doi: 10.1016/j.indcrop.2025.122270. Full text in Research Archive Show summary

  Shiitake mushroom cultivation could effectively reduce the recalcitrance of hardwood, facilitating cellulosic ethanol production. However, the shiitake pretreated spent mushroom substrates (SMS) have exhibited significant variations in enzymatic saccharification and fermentation efficiencies in previous studies. Parallel with shiitake cultivation, significant increases in extractives mass were observed in the SMS. This highlights the need to examine the formation of pretreatment by-products and their potential impact on downstream bioconversion processes. In this study, shiitake cultivation resulted in 42.6–47.6 % degradation of lignocellulosic components, with continuous generation of various small molecules. Shiitake demonstrated extensive and selective utilization of these degradation products during fructification. Consequently, non-utilized molecules, including (L)-dehydroascorbic acid, triglochinic acid, and 5-hydroxy-2-methylchromone, accumulated in SMS extractives as pretreatment by-products. The SMS cellulose showed 58.8 % digestibility upon enzymatic saccharification. The resulting hydrolysate was fermented to ethanol by Saccharomyces cerevisiae rendering 83.4 % of the theoretical yield. Although water extraction effectively minimized the by-products accumulation in SMS and hydrolysate, no improvement of enzymatic saccharification and fermentation efficiencies were observed. In contrast to thermochemical pretreatment methods, the by-products derived from shiitake pretreatment had limited inhibitory effects on downstream bioconversion processes. This study provides valuable indications for further optimization of shiitake pretreatment towards industrial implementation.

• Akter, Afrina; Klausen, Sarah Jasmin; Soto, Luis Alejandro Romero; Díaz, Francisco; Domínguez, Herminia & Xiong, Shaojun [Show all 8 contributors for this article] (2025). Comparative extraction of bioactive compounds from spent mushroom substrates of Lentinula edodes and Pleurotus ostreatus using subcritical water and pressurized ethanol. Industrial crops and products (Print). ISSN 0926-6690. 235. doi: 10.1016/j.indcrop.2025.121750. Full text in Research Archive Show summary

  This study aimed to assess subcritical water extraction (SWE) and pressurized ethanol extraction (PEE) for recovering bioactive compounds from spent mushroom substrate (SMS) derived from shiitake and oyster mushrooms. The extraction of bioactive compounds can facilitate the valorisation of SMS, an increasingly important waste stream whose management and disposal pose challenges for both mushroom producers and the environment. A 32 experimental design, using three temperatures (125, 150, and 175 °C) and three holding times (0, 15, and 30 min) was used in SWE. PEE was run at a single temperature (175 °C) and two holding times (0 and 15 min). The experimental results revealed that the highest extraction yield was achieved in shiitake SMS using SWE at 175 °C and 15 min. In most SWE conditions, shiitake SMS yielded higher concentrations of total phenolic compounds, total carbohydrates, and β-glucans compared to oyster mushroom SMS. Ascorbic acid, vanillic acid, protocatechuic acid, and gallic acid were the most abundant phenolic acids identified in both extracts. The SWE extracts of shiitake SMS exhibited higher DPPH and FRAP antioxidant activity than those of oyster mushroom SMS, and this activity increased with increasing temperature and time. PEE extracts exhibited higher DPPH and TEAC antioxidant activity for oyster mushroom SMS compared to shiitake SMS and showed a negative trend with increasing extraction time at 175 °C for both SMS. Our results show that SWE and PEE are viable methods for extracting bioactive compounds from SMS.

• Quisth, Ida; Ulfsparre, Isabella; Müller, Bettina; Passoth, Volkmar; Solberg, Svein Øivind & Martín, Carlos [Show all 9 contributors for this article] (2025). Spent Mushroom Substrate (SMS) as a Sustainable Soil Amendment and Biofertilizer: A Review of Opportunities and Challenges in Agricultural and Horticultural Systems. Agricultural and Food Science. ISSN 1459-6067. 34(4), p. 323–338. doi: 10.23986/afsci.163895. Full text in Research Archive Show summary

  Spent mushroom substrate (SMS), a byproduct of mushroom cultivation, has gained increasing attention as a sustainable soil amendment in agricultural and horticultural systems. SMS provides a promising alternative to peat-based substrates and synthetic fertilizers, supporting circular bioeconomy principles and sustainable farming practices. This is particularly relevant in boreal regions, where soils are typically acidic, nutrient-poor, and subject to short growing season conditions, presenting both opportunities and challenges for organic inputs. This review synthesizes current research on the agronomic and environmental implications of SMS use in agriculture and horticulture, including its effects on seed germination, plant growth, crop yield, root development, soil structure, and disease suppression. From an environmental perspective, the use of SMS contributes to waste reduction by repurposing organic residues and replacing peat, a non-renewable resource with significant ecological costs. However, several hurdles remain, including high salinity, inconsistent composition, nutrient imbalances, and complex compounds difficult for plants to access, which can impede plant performance. While prior studies have explored SMS in isolated settings, a comprehensive evaluation across systems is lacking. This review addresses that gap by assessing current evidence, identifying limitations, and outlining future research needs to optimize the use of SMS and scale its adoption in sustainable farming.

• Klausen, Sarah Jasmin; Soto, Luis Alejandro Romero; Liaqat, Ayesha; Dehghanmanshadi, Zahra; Strætkvern, Knut Olav & Xiong, Shaojun [Show all 7 contributors for this article] (2025). Biorefining spent substrates of shiitake (Lentinula edodes) and oyster mushroom (Pleurotus ostreatus): enzymatic saccharification of cellulose and xylan, with lignin recovery from residues. Bioengineered. ISSN 2165-5979. 16(1). doi: 10.1080/21655979.2025.2536443. Full text in Research Archive Show summary

  Spent mushroom substrate (SMS), the main by-product of mushroom cultivation, is a source of sugars that can be released by saccharification. This work aimed at investigating the enzymatic saccharification of the polysaccharides of the SMS of shiitake (Lentinula edodes) and oyster mushroom (Pleurotus ostreatus) and exploring the lignin extraction from the saccharification residues. First, analytical enzymatic saccharification (AES) with a cellulase cocktail and an experimental hemicellulase-rich preparation was applied. AES revealed higher digestibility of both polysaccharides for shiitake SMS than for oyster mushroom SMS. Using the cellulase cocktail, shiitake SMS resulted in a digestibility above 80% and 70% (w/w) for cellulose and xylan, respectively, while the maximum values for oyster mushroom SMS were 52% and 32% (w/w). The experimental enzyme preparation resulted in lower cellulose digestibility and higher xylan digestibility. Still, the saccharification trend between the two SMS types remained unchanged. To enhance the enzymatic saccharification of oyster mushroom SMS, hydrothermal treatment was applied. The treatment improved the enzymatic digestibility of cellulose by up to 84%. A validation experiment at larger scale showed that hydrothermally treated oyster mushroom SMS had a comparable overall conversion with non-treated shiitake SMS. Following a biorefinery strategy, lignin was extracted from the residues of the preparative enzymatic saccharification using the green solvent γ-valerolactone under different temperatures and holding times. The extracted product contained 98.8% lignin and did not contain cellulose or xylan. The results of this study provide the grounds for biorefinery processes enabling recovery of bioactive compounds, fermentable sugars, and high-quality lignin from SMS

• Serrano-Febles, Jonathan; Luis-Orozco, Jesús; Ramírez, Héctor Luis; Gómez-Brizuela, Leissy; Martín, Carlos & García-Cruz, Ariel (2025). Optimization of enzymatic hydrolysis of corn starch to obtain glucose syrups by genetic algorithm. Dyna. ISSN 0012-7353. 92(235), p. 83–91. doi: 10.15446/dyna.v92n235.116302. Full text in Research Archive Show summary

  This work corresponds to the optimization of the operating variables of the enzymatic hydrolysis of corn starch to obtain glucose syrups using the genetic algorithm of Matlab (2020a). For this reason, the hydrolytic process is mathematically modeled by response surface methodology. Pareto chart indicated that saccharification variables exert the highest influence on starch conversion. This mathematical model is beneficial for a better understanding and operational control of hydrolysis at the industrial level. The optimization problem solution shows that a maximum dextrose equivalent of 98.13% can be reached if the hydrolysis is performed under optimal operating conditions, which were also confirmed experimentally. The results show that to achieve the highest yield, liquefaction should be carried out at a temperature of 92oC, pH of 6.3, α-amylase dose of 1.5 mg enzyme/g starch and hydrolysis time of 1 hour; while saccharification should be conducted at a temperature of 57oC, pH of 4.9, glucoamylase dose of 1.15 mg enzyme/g starch and hydrolysis time of 34 hours. The reversion phenomenon is detected when the hydrolysis time exceeds 35 hours, with a negative incidence on the dextrose equivalent.

• Semaan, Georgeio; Klausen, Sarah Jasmin; Martín, Carlos; Jørgensen, Kåre Bredeli & Kumar, Gopalakrishnan (2025). Fractionation and characterization of multi-feedstock lignocellulosic biomass via two-stage pretreatment. Biomass Conversion and Biorefinery. ISSN 2190-6815. 15, p. 18925–18945. doi: 10.1007/s13399-025-06576-8. Full text in Research Archive Show summary

  A two-stage pretreatment toward lignocellulosic biomass fractionation was devised. The process consisted of dilute acid hydrolysis using oxalic acid, followed by oxalic acid–assisted ethanol organosolv pretreatment. A biomass mixture consisting of four regional lignocellulosic materials, namely, brewer’s spent grain, tomato waste biomass, cucumber waste biomass, and spent coffee grounds, was used. In the first stage, the optimum mixture composition was determined using a full factorial design coupled with a simplex-centroid design. The interactive effects of the solid-to-liquid ratio, holding time, acid type, and concentration were also considered. In the attempt to lower solid yields and increase hemicellulose dissolution, elevated levels of furfural (15.73 g/L) and 5-hydroxymethylfurfural (8.56 g/L) were formed, due to increased pretreatment severity (180 min, 135C, 83.15 mg oxalic acid/g biomass, and 100 g biomass/L). The solid yield achieved was 50.53%. In the second stage, the effect of ethanol-to-water solvent ratio, holding time, and temperature were investigated using a central composite experimental design. Solid yields ranged between 72.57 and 85.20% (w/w), mainly due to lignin removal. Pretreatment with 75% (v/v) ethanol at 120 min and 190 °C resulted in the highest lignin recovery (44.69%). Post-experimental verification runs were performed to evaluate the validity of the response surface models with a maximum error of 15.17%. Characterization by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and heteronuclear single quantum coherence spectroscopy (HSQC) were conducted to assess biomass fraction integrity and structural changes during pretreatment.

• Chen, Feng; Martín, Carlos; Jönsson, Leif J.; Gandla, Madhavi Latha; Klausen, Sarah Jasmin & Soto, Luis Alejandro Romero [Show all 7 contributors for this article] (2024). Mild thermal treatment assists fungal preprocessing of softwood sawdust for production of fermentable sugar. Industrial crops and products (Print). ISSN 0926-6690. 223. doi: 10.1016/j.indcrop.2024.120284. Full text in Research Archive Show summary

  Preheating with hot air at 85 – 125 ◦ ◦ C was evaluated for its effectiveness in removing terpenes and terpenoids in softwood sawdust, thereby enhancing fungal preprocessing and subsequent saccharification of softwood-based mushroom substrates. Sawdust from pine (Pinus sylvestris L.) and spruce (Picea abies (L.) H. Karst.) was preheated prior to shiitake (Lentinula edodes (Berk.) Pegler) cultivation. Preheating removed up to 96 % of terpenes in pine- based substrates and up to 50 % in spruce-based substrates. Additionally, preheating decreased total terpenoids content in spruce by up to 78 %. For the pine-based substrate, the mild heating generally led to faster colonisation and improved mushroom yield, with the fastest mycelia colonisation and highest yield observed for 105 C treatment. This temperature was associated with the lowest content of total terpenes and absence of major monoterpenes. The content of terpenes and terpenoids continued to decrease during cultivation, alongside fungal degradation of lignocellulose. As a result of more extensive lignin degradation, the enzymatic digestibility of cellulose was higher for spruce-based spent mushroom substrate than for pine-based one (up to 89 % vs. 49 % conversion). Enzymatic digestibility showed a negative correlation with the α correlation with increasing preheating temperatures.

• Rodrigues, Danielle Matias; Silva, Marcos Fellipe da; Almeida, Francisco Lucas Chaves; Mélo, Allan Henrique Félix de; Forte, Marcus Bruno Soares & Martín, Carlos [Show all 9 contributors for this article] (2024). A green approach to biomass residue valorization: Bacterial nanocellulose production from agro-industrial waste. Biocatalysis and Agricultural Biotechnology. ISSN 1878-8181. 56. doi: 10.1016/j.bcab.2024.103036. Full text in Research Archive
• Silva, Vinicius Fernandes Nunes da; Menezes, Fabricia Farias de; Gonçalves, Adilson Roberto; Martín, Carlos & Rocha, George Jackson de Moraes (2024). Modulating the properties and structure of lignins produced by alkaline delignification of sugarcane bagasse pretreated with two different mineral acids at pilot-scale. International Journal of Biological Macromolecules. ISSN 0141-8130. 263(1). doi: 10.1016/j.ijbiomac.2024.130111. Full text in Research Archive
• González-Gloria, Karla D.; Tomás-Pejó, Elia; Amaya-Delgado, Lorena; Rodríguez-Jasso, Rosa M.; Loredo-Treviño, Araceli & Singh, Anusuiya [Show all 10 contributors for this article] (2024). Biochemical and Biorefinery Platform for Second-Generation Bioethanol: Fermentative Strategies and Microorganisms. Fermentation. 10(7). doi: 10.3390/fermentation10070361. Full text in Research Archive Show summary

  Bioethanol is the most commonly used biofuel. It is an alternative to replace fossil fuels in renewable energy; it can be produced from lignocellulosic feedstock using a biotechnological process. Their participation of microorganisms is crucial in the bioconversion process of fermentation for ethanol production and can involve bacteria, fungi, and yeasts. However, when working within bioethanol processes from lignocellulose feedstock, microorganisms face some challenges, such as high temperature, high solids content, and the ability to ferment sugars for high ethanol concentration. Such challenges will depend on operative strategies, such as simultaneous saccharification and fermentation, separate hydrolysis and fermentation, semi-simultaneous saccharification and fermentation, and consolidated bioprocessing; these are the most common configurations. This review presents different trends of the microbial role, biochemical application, and fermentation operative strategies for bioethanol production of the second generation.

• Berglund, Linn; Völtz, Luisa Rosenstock; Gehrmann, Timon; Antonopoulou, Io; Cristescu, Carmen & Xiong, Shaojun [Show all 10 contributors for this article] (2024). The use of spent mushroom substrate as biologically pretreated wood and its fibrillation. Journal of Environmental Management (JEM). ISSN 0301-4797. 372. doi: 10.1016/j.jenvman.2024.123338. Full text in Research Archive Show summary

  Utilization of biomass and reuse of industrial by-products and their sustainable and resource-efficient development into products that are inherently non-toxic is important to reduce the use of hazardous substances in the design, manufacture and application of biomaterials. The hypothesis in this study is that spent mushroom substrate (SMS), a by-product from mushroom production, has already undergone a biological pretreatment and thus, can be used directly as a starting material for fibrillation into value-added and functional biomaterial, without the use of toxic substances. The study show that SMS can be effectively fibrillated at a very high concentration of 6.5 wt % into fibrils using an energy demand of only 1.7 kWh kg−1, compared to commercial and chemically pretreated wood pulp at 8 kWh kg−1, under same processing conditions. SMS is a promising resource for fibrillation with natural antioxidant activity and network formation ability, which are of interest to explore further in applications such as packaging. The study shows that biological pretreatment can offer lower environmental impact related to toxic substances emitted to the environment and thus contribute to reduced impacts on categories such as water organisms, human health, terrestrial organisms, and terrestrial plants compared to chemical pretreatments.

• Klausen, Sarah Jasmin; Falck-Ytter, Anne Bergljot; Strætkvern, Knut Olav & Martín, Carlos (2023). Evaluation of the Extraction of Bioactive Compounds and the Saccharification of Cellulose as a Route for the Valorization of Spent Mushroom Substrate. Molecules. ISSN 1431-5157. 28(13). doi: 10.3390/molecules28135140. Full text in Research Archive Show summary

  The extraction of bioactive compounds and cellulose saccharification are potential directions for the valorization of spent mushroom substrate (SMS). Therefore, investigating the suitability of different extraction methods for recovering bioactive compounds from SMS and how the extractionmaffects the enzymatic saccharification is of uppermost relevance. In this work, bioactive compounds were extracted from Pleurotus spp. SMS using four extraction methods. For Soxhlet extraction (SoE), a 40:60 ethanol/water mixture gave the highest extraction efficiency (EE) (69.9–71.1%) among the seven solvent systems assayed. Reflux extraction with 40:60 ethanol/water increased the extraction yield and EE compared to SoE. A shorter reflux time yielded a higher extraction of carbohydrates than SoE, while a longer time was more effective for extracting phenolics. The extracts from 240 min of reflux had comparable antioxidant activity (0.3–0.5 mM GAE) with that achieved for SoE. Ultrasound-assisted extraction (UAE) at 65 ◦C for 60 min allowed an EE (~82%) higher than that achieved by either reflux for up to 150 min or SoE. Subcritical water extraction (SWE) at 150 ◦C resulted in the best extraction parameters among all the tested methods. Vanillic acid and chlorogenic acid were the primary phenolic acids identified in the extracts. A good correlation between the concentration of caffeic acid and the antioxidant activity of the extracts was found. Saccharification tests revealed an enhancement of the enzymatic digestibility of SMS cellulose after the extraction of bioactive compounds. The findings of this initial study provide indications on new research directions for maximizing the recovery of bioactive compounds and fermentable sugars from SMS.

• Miranda, Diego A.; Marín, Katherine; Sundman, Ola; Hedenström, Mattias; Quillaguaman, Jorge & Gorzsás, András [Show all 13 contributors for this article] (2023). Production and Characterization of Poly(3-hydroxybutyrate) from Halomonas boliviensis LC1 Cultivated in Hydrolysates of Quinoa Stalks. Fermentation. 9(6). doi: 10.3390/fermentation9060556. Full text in Research Archive Show summary

  The global production of fossil-based plastics has reached critical levels, and their substitution with bio-based polymers is an urgent requirement. Poly(3-hydroxybutyrate) (PHB) is a biopolymer that can be produced via microbial cultivation, but efficient microorganisms and low-cost substrates are required. Halomonas boliviensis LC1, a moderately halophilic bacterium, is an effective PHB producer, and hydrolysates of the residual stalks of quinoa (Chenopodium quinoa Willd.) can be considered a cheap source of sugars for microbial fermentation processes in quinoa-producing countries. In this study, H. boliviensis LC1 was adapted to a cellulosic hydrolysate of quinoa stalks obtained via acid-catalyzed hydrothermal pretreatment and enzymatic saccharification. The adapted strain was cultivated in hydrolysates and synthetic media, each of them with two different initial concentrations of glucose. Cell growth, glucose consumption, and PHB formation during cultivation were assessed. The cultivation results showed an initial lag in microbial growth and glucose consumption in the quinoa hydrolysates compared to cultivation in synthetic medium, but after 33 h, the values were comparable for all media. Cultivation in hydrolysates with an initial glucose concentration of 15 g/L resulted in a higher glucose consumption rate (0.15 g/(L h) vs. 0.14 g/(L h)) and volumetric productivity of PHB (14.02 mg/(L h) vs. 10.89 mg/(L h)) than cultivation in hydrolysates with 20 g/L as the initial glucose concentration. During most of the cultivation time, the PHB yield on initial glucose was higher for cultivation in synthetic medium than in hydrolysates. The produced PHBs were characterized using advanced analytical techniques, such as high-performance size-exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). HPSEC revealed that the molecular weight of PHB produced in the cellulosic hydrolysate was lower than that of PHB produced in synthetic medium. TGA showed higher thermal stability for PHB produced in synthetic medium than for that produced in the hydrolysate. The results of the other characterization techniques displayed comparable features for both PHB samples. The presented results show the feasibility of producing PHB from quinoa stalks with H. boliviensis.

• Martín, Carlos; Xiong, Shaojun; Passoth, Volkmar; Põldmaa, Kadri; Solberg, Svein Øivind & Hultberg, M [Show all 13 contributors for this article] (2023). Mushrooms for enhanced agriculture sustainability the MUSA concept. C3-Bioeconomy Circular and Sustainable Bioeconomy. p. 131–146. Full text in Research Archive Show summary

  The project MUSA – MUshrooms for Sustainable Agriculture is an effort to use mushroom-based processes to enhance agriculture sustainability in Nordic and Baltic countries. The project covers both the production of fruitbodies of edible fungi and the upgrading of the exhausted substrate from mushroom cultivation. The suitability of residues generated locally for producing edible mushrooms is investigated. Residues from Nordic agriculture and sub-utilized streams from forestry management, as well as wood processing by-products, are evaluated as the substrate base for producing shiitake (Lentinula edodes) and oyster (Pleurotus spp.) mushrooms. The project explores the potential of spent mushroom substrate (SMS) to support food production. SMS prospective as source of bioactive compounds and sugars is evaluated. MUSA investigates the suitability of SMS hydrolysates as carbon sources for cultivating oleaginous yeast to produce microbial oil suitable for human consumption. Using SMS for substituting mineral fertilizers and providing wastewater bioremediation solutions is also assessed

• Castro, Eulogio; Strætkvern, Knut Olav; Romero-García, J.M. & Martín, Carlos (2023). Pretreatment and Bioconversion for Valorization of Residues of Non-Edible Oilseeds. Agronomy. 13(9). doi: 10.3390/agronomy13092196. Full text in Research Archive Show summary

  Biodiesel production currently follows a first-generation model using edible oils as raw materials. Such a production model is unsustainable, considering that it is limited by the high cost of edible oils, competes with the food sector, and is linked to deforestation and other environmental threats. Changing the raw material base to non-edible oils provides an opportunity to increase the sustainability of the biodiesel industry and to avoid conflicts with food production. Processing non-edible oilseeds for extracting the oil to be used for producing biodiesel generates large amounts of residues, such as de-oiled cakes, seed husks, and fruit shells and pods as well as plant stems and leaves resulting from pruning and other agronomy practices. Most of those residues are currently disposed of by burning or used in a suboptimal way. Bioconversion following the sugar platform route, anaerobic digestion, or enzyme production provides means for upgrading them to advanced biofuels and high-added value products. Bioconversion of plant biomass, including oilseed residues, requires pretreatment to enhance their susceptibility to enzymes and microorganisms. This review provides an outlook on bioconversion approaches applicable to different residues of oilseed-bearing plant species. Recent reports on the pretreatment of non-edible oilseed residues for enhancing their bioconversion through either the sugar platform route or anaerobic digestion are critically discussed. This review is based on an exhaustive Web of Science search performed in January–May 2023.

• Martín, Carlos; Zervakis, Georgios I.; Xiong, Shaojun; Koutrotsios, Georgios & Strætkvern, Knut Olav (2023). Spent substrate from mushroom cultivation: exploitation potential toward various applications and value-added products. Bioengineered. ISSN 2165-5979. 14(1), p. 2252138–2252138. doi: 10.1080/21655979.2023.2252138. Full text in Research Archive Show summary

  Spent mushroom substrate (SMS) is the residual biomass generated after harvesting the fruitbodies of edible/medicinal fungi. Disposal of SMS, the main by-product of the mushroom cultivation process, often leads to serious environmental problems and is financially demanding. Efficient recycling and valorization of SMS are crucial for the sustainable development of the mushroom industry in the frame of the circular economy principles. The physical properties and chemical composition of SMS are a solid fundament for developing several applications, and recent literature shows an increasing research interest in exploiting that inherent potential. This review provides a thorough outlook on SMS exploitation possibilities and discusses critically recent findings related to specific applications in plant and mushroom cultivation, animal husbandry, and recovery of enzymes and bioactive compounds.

• Chen, Feng; Medina, Carlos Orestes Martin; Lestander, Torbjörn A.; Grimm, Alejandro & Xiong, Shaojun (2022). Shiitake cultivation as biological preprocessing of lignocellulosic feedstocks – Substrate changes in crystallinity, syringyl/guaiacyl lignin and degradation-derived by-products. Bioresource Technology. ISSN 0960-8524. 344. doi: 10.1016/j.biortech.2021.126256. Full text in Research Archive Show summary

  Formulation of substrates based on three hardwood species combined with modulation of nitrogen content by whey addition (0–2%) was investigated in an experiment designed in D-optimal model for their effects on biological preproceesing of lignocellulosic feedstock by shiitake mushroom (Lentinula edodes) cultivation. Nitrogen loading was shown a more significant role than wood species for both mushroom production and lignocellulose degradation. The fastest mycelial colonisation occurred with no nitrogen supplementation, but the highest mushroom yields were achieved when 1% whey was added. Low nitrogen content resulted in increased delignification and minimal glucan consumption. Delignification was correlated with degradation of syringyl lignin unit, as indicated by a significant reduction (41.5%) of the syringyl-to-guaiacyl ratio after cultivation. No significant changes in substrate crystallinity were observed. The formation of furan aldehydes and aliphatic acids was negligible during the pasteurisation and fungal cultivation, while the content of soluble phenolics increased up to seven-fold.

• Medina, Carlos Orestes Martin; Carrasco, Cristhian; Jönsson, Leif J.; Romero-Soto, Luis; Chambi, Diego & Oliva-Taravilla, A (2022). Biorefining of quinoa residues for production of advanced biofuels and biopolymers. European biomass conference and exhibition proceedings. doi: 10.5071/30thEUBCE2022-6CV.9.1. Full text in Research Archive
• Chen, Feng; Medina, Carlos Orestes Martin; Finell, Michael & Xiong, Shaojun (2022). Enabling efficient bioconversion of birch biomass by Lentinula edodes: regulatory roles of nitrogen and bark additions on mushroom production and cellulose saccharification. Biomass Conversion and Biorefinery. ISSN 2190-6815. 12, p. 1217–1227. doi: 10.1007/s13399-020-00794-y. Full text in Research Archive Show summary

  Pretreatment with edible white-rot fungi has advantages in low inputs of energy and chemicals for reducing the recalcitrance of woody biomass for bioethanol production while harvesting protein-rich food. The effectiveness of fungal pretreatment may vary with substrate composition. In this study, birch with or without bark and nitrogen additives were experimentally studied for their effects on shiitake production, substrate lignocellulosic degradation and enzymatic convertibility with cellulolytic enzymes. Whey was added as protein nitrogen and led to successful outcomes, while non-protein nitrogen urea and ammonium-nitrate resulted in mortality of fungal mycelia. The mushroom yields of one harvest were generally comparable between the treatments, averaging 651 g fresh weight per kilogram dry substrate, and high enough as to be profitable. Nitrogen loading (0.5–0.8%, dry mass) negatively affected lignin degradation and enzymatic convertibility and prolonged cultivation/pretreatment time. The added bark (0–20%) showed quadratic correlation with degradation of lignin, xylan and glucan as well as enzymatic digestibility of glucan. Nitrogen loading of < 0.6% led to maximal mass degradation of xylan and lignin at bark ratios of 4–9% and 14–19%, respectively, peak saccharification of glucan at 6–12% and the shortest pretreatment time at 8–13% bark. The designed substrates resulted in 19–35% of glucan mass loss after fungal pretreatment, less than half of the previously reported values. Nitrogen and bark additions can regulate lignocellulose degradation and saccharification of birch-based substrates. The designed substrate composition could considerably reduce cellulose consumption during fungal pretreatment, thus improving bioconversion efficiency.

• Martín, Carlos; Dixit, Pooja; Momayez, Forough & Jönsson, Leif J. (2022). Hydrothermal pretreatment of lignocellulosic feedstocks to facilitate biochemical conversion. Frontiers in Bioengineering and Biotechnology. 10. doi: 10.3389/fbioe.2022.846592. Full text in Research Archive Show summary

  Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other biobased commodities typically includes physical diminution, hydrothermal pretreatment, enzymatic saccharification, and valorization of sugars and hydrolysis lignin. This approach is also known as a sugar-platform process. The goal of the pretreatment is to facilitate the ensuing enzymatic saccharification of cellulose, which is otherwise impractical due to the recalcitrance of lignocellulosic feedstocks. This review focuses on hydrothermal pretreatment in comparison to alternative pretreatment methods, biomass properties and recalcitrance, reaction conditions and chemistry of hydrothermal pretreatment, methodology for characterization of pretreatment processes and pretreated materials, and how pretreatment affects subsequent process steps, such as enzymatic saccharification and microbial fermentation. Biochemical conversion based on hydrothermal pretreatment of lignocellulosic feedstocks has emerged as a technology of high industrial relevance and as an area where advances in modern industrial biotechnology become useful for reducing environmental problems and the dependence on fossil resources.

• Momayez, Forough; Hedenström, Mattias; Stagge, Stefan; Jönsson, Leif & Martín, Carlos (2022). Valorization of hydrolysis lignin from a spruce-based biorefinery by applying γ-valerolactone treatment. Bioresource Technology. ISSN 0960-8524. 359. doi: 10.1016/j.biortech.2022.127466. Full text in Research Archive Show summary

  Hydrolysis lignin, i.e., the hydrolysis residue of cellulosic ethanol plants, was extracted with the green solvent γ-valerolactone (GVL). Treatments at 170–210 ◦C were performed with either non-acidified GVL/water mixtures (NA-GVL) or with mixtures containing sulfuric acid (SA-GVL). SA-GVL treatment at 210 ◦C resulted in the highest lignin solubilization (64% (w/w) of initial content), and 76% of the solubilized mass was regenerated by water induced precipitation. Regenerated lignins were characterized through compositional analysis with sulfuric acid, as well as using pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS), high-performance size-exclusion chromatography (HPSEC), solid-state cross-polarization/magic angle spinning 13C nuclear magnetic resonance (CP/MAS 13C NMR) spectroscopy, 1 H–13C heteronuclear single-quantum coherence NMR (HSQC NMR), and Fourier-transform infrared (FTIR) spectroscopy. The characterization revealed that the main difference between regenerated lignins was their molecular weight. Molecular weight averages increased with treatment temperature, and they were higher and had broader distribution for SA-GVL lignins than for NA-GVL lignins.

• Chambi, Diego; Lundqvist, Jenny; Nygren, Erik; Romero-Soto, Luis; Marín, Katherine & Gorzsás, András [Show all 13 contributors for this article] (2022). Production of exopolysaccharides by cultivation of halotolerant Bacillus atrophaeus BU4 in glucose- and xylose-based synthetic media and in hydrolysates of quinoa stalks. Fermentation. 8(2). doi: 10.3390/fermentation8020079. Full text in Research Archive Show summary

  A halotolerant, exopolysaccharide-producing bacterium isolated from the Salar de Uyuni salt flat in Bolivia was identified as Bacillus atrophaeus using next-generation sequencing. Comparisons indicate that the genome most likely (p-value: 0.0024) belongs to a subspecies previously not represented in the database. The growth of the bacterial strain and its ability to produce exopolysaccharides (EPS) in synthetic media with glucose or xylose as carbon sources, and in hydrolysates of quinoa stalks, was investigated. The strain grew well in all synthetic media, but the growth in glucose was better than that in xylose. Sugar consumption was better when initial concentrations were low. The growth was good in enzymatically produced cellulosic hydrolysates but was inhibited in hemicellulosic hydrolysates produced using hydrothermal pretreatment. The EPS yields were up to 0.064 g/g on initial glucose and 0.047 g/g on initial xylose, and was higher in media with relatively low sugar concentrations. The EPS was isolated and purified by a sequential procedure including centrifugation, cold ethanol precipitation, trichloroacetic acid treatment, dialysis, and freeze-drying. Glucose and mannose were the main sugars identified in hydrolyzed EPS. The EPS was characterized by size-exclusion chromatography, Fourier-transform infrared (FTIR) spectroscopy, heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis. No major differences were elucidated between EPS resulting from cultivations in glucose- or-xylose-based synthetic media, while some divergences with regard to molecular-weight averages and FTIR and HSQC NMR spectra were detected for EPS from hydrolysate-based media.

• Chen, Feng; Xiong, Shaojun; Gandla, Madhavi Latha; Stagge, Stefan & Medina, Carlos Orestes Martin (2021). Spent mushroom substrates for ethanol production – Effect of chemical and structural factors on enzymatic saccharification and ethanolic fermentation of Lentinula edodes-pretreated hardwood. Bioresource Technology. ISSN 0960-8524. 347, p. 1–12. doi: 10.1016/j.biortech.2021.126381. Full text in Research Archive Show summary

  Spent mushroom substrates (SMS) from cultivation of shiitake (Lentinula edodes) on three hardwood species were investigated regarding their potential for cellulose saccharification and for ethanolic fermentation of the produced hydrolysates. High glucan digestibility was achieved during enzymatic saccharification of the SMSs, which was related to the low mass fractions of lignin and xylan, and it was neither affected by the relative content of lignin guaiacyl units nor the substrate crystallinity. The high nitrogen content in SMS hydrolysates, which was a consequence of the fungal pretreatment, was positive for the fermentation, and it ensured ethanol yields corresponding to 84–87% of the theoretical value in fermentations without nutrient supplementation. Phenolic compounds and acetic acid were detected in the SMS hydrolysates, but due to their low concentrations, the inhibitory effect was limited. The solid leftovers resulting from SMS hydrolysis and the fermentation residues were quantified and characterized for further valorisation.

• Flores-Copa, V; Romero-Soto, Luis; Romero-Calle, D; Alvarez-Aliaga, MT; Orozco-Gutiérrez, F & Vega-Baudrit, J [Show all 8 contributors for this article] (2021). Residual brewing yeast as substrate for co-production of cell biomass and biofilm using Candida maltosa SM4. Fermentation. 72(2). doi: 10.3390/fermentation7020084. Full text in Research Archive
• Carrasco, Cristhian; Jönsson, Leif J. & Martín, Carlos (2021). Hydrothermal pretreatment of water-extracted and aqueous-ethanol-extracted quinoa stalks for enzymatic saccharification of cellulose. Energies. 14(14). doi: 10.3390/en14144102. Full text in Research Archive
• Ilanidis, Dimitrios; Stagge, Stefan; Jönsson, Leif J. & Martín, Carlos (2021). Hydrothermal pretreatment of wheat straw: effects of temperature and acidity on by-product formation and inhibition of enzymatic hydrolysis and ethanolic fermentation. Agronomy. 11(3). doi: 10.3390/agronomy11030487. Full text in Research Archive
• Chambi, Diego; Romero-Soto, Luis; Villca, R; Orozco-Gutiérrez, F; Vega-Baudrit, J & Quillaguaman, Jorge [Show all 9 contributors for this article] (2021). Exopolysaccharides production by cultivating a bacterial isolate from the hypersaline environment of Salar de Uyuni (Bolivia) in pretreatment liquids of steam-exploded quinoa stalks and enzymatic hydrolysates of Curupaú sawdust. Fermentation. 7(1). doi: 10.3390/fermentation7010033. Full text in Research Archive
• Martín, Carlos; Ilanidis, Dimitrios; Stagge, Stefan & Jönsson, Leif J. (2021). Effects of operational conditions on auto-catalyzed and sulfuric-acid-catalyzed hydrothermal pretreatment of sugarcane bagasse at different severity factor. Industrial crops and products (Print). ISSN 0926-6690. 159. doi: 10.1016/j.indcrop.2020.113077. Full text in Research Archive
• Martín, Carlos (2021). Pretreatment of crop residues for bioconversion. Agronomy. 11(5). doi: 10.3390/agronomy11050924. Full text in Research Archive
• Martín, Carlos; Ilanidis, Dimitrios; Wu, Guochao; Stagge, Stefan & Jönsson, Leif J. (2021). Effects of redox environment on hydrothermal pretreatment of lignocellulosic biomass under acidic conditions. Bioresource Technology. ISSN 0960-8524. 319. doi: 10.1016/j.biortech.2020.124211. Full text in Research Archive
• Chen, Feng; Grimm, Alejandro; Eilertsen, L.; Martín, Carlos; Arshadi, Mehrdad & Xiong, Shaojun (2021). Integrated production of edible mushroom (Auricularia auricular-judae), fermentable sugar and solid biofuel. Renewable Energy. ISSN 0960-1481. 170, p. 172–180. doi: 10.1016/j.renene.2021.01.124. Full text in Research Archive
• Chacón-Navarrete, Helena; Medina, Carlos Orestes Martin & Moreno-García, Jaime (2021). Yeast immobilization systems for second-generation ethanol production: actual trends and future perspectives. Biofuels, Bioproducts and Biorefining. ISSN 1932-104X. 15(5), p. 1549–1565. doi: 10.1002/bbb.2250. Full text in Research Archive Show summary

  Yeast immobilization with low-cost carrier materials is a suitable strategy to optimize the fermentation of lignocellulosic hydrolysates for the production of second-generation (2G) ethanol. It is defined as the physical confinement of intact cells to a certain region of space (the carrier) with the preservation of their biological activity. This technological approach facilitates promising strategies for second-generation bioethanol production due to the enhancement of the fermentation performance that is expected to be achieved. Using immobilized cells, the resistance to inhibitors contained in the hydrolysates and the co-utilization of sugars are improved, along with facilitating separation operations and the reuse of yeast in new production cycles. Until now, the most common immobilization technology used calcium alginate as a yeast carrier but other supports such as biochar or multispecies biofilm membranes have emerged as interesting alternatives. This review compiles updated information about cell carriers and yeast-cell requirements for immobilization, and the benefits and drawbacks of different immobilization systems for second-generation bioethanol production are investigated and compared. © 2021 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.

• Rocha, GJM; Andrade, L; Martín, Carlos; Araujo, GT; Filho, V & Curvelo, A (2020). Simultaneous obtaining of oxidized lignin and cellulosic pulp from steam-exploded sugarcane bagasse. Industrial crops and products (Print). ISSN 0926-6690. 147. Full text in Research Archive
• Oliva-Taravilla, A; Carrasco, Carlos; Jönsson, Leif J. & Martín, Carlos (2020). Effects of biosurfactants on enzymatic saccharification and fermentation of pretreated softwood. Molecules. ISSN 1431-5157. 25(16). doi: 10.3390/molecules25163559. Full text in Research Archive
• Chen, Feng; Xiong, Shaojun; Sundelin, J; Martín, Carlos & Hultberg, M (2020). Potential for combined production of food and biofuel: cultivation of Pleurotus pulmonarius on soft- and hardwood sawdusts. Journal of Cleaner Production. ISSN 0959-6526. 266. Full text in Research Archive

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• Martín, Carlos (2021). Pretreatment and bioconversion of crop residues. MDPI. ISBN 9783036514109. 248 p. Full text in Research Archive

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• E., Velasco-Zambrano,; E., Espinosa,; E., Castro,; J.M., Romero-García,; J, Moreno, & Martín, Carlos [Show all 8 contributors for this article] (2025). Fungal pellet immobilization of modified Escherichia coli for enhanced bioethanol production from red grape stalks. Full text in Research Archive
• D.A., Miranda,; Soto, Luis Alejandro Romero; L.J., Jönsson,; C., Carrasco, & Martín, Carlos (2025). Sustainable production of biopolymers from underexplored agro-waste using halophilic and halotolerant bacteria. Full text in Research Archive
• D.A., Miranda,; Soto, Luis Alejandro Romero; L.J., Jönsson,; C., Carrasco, & Martín, Carlos (2025). Sustainable production of biopolymers from underexplored agro-waste using halotolerant bacteria. Full text in Research Archive
• Klausen, Sarah Jasmin; Akter, Afrina; Soto, Luis Alejandro Romero; Martín, Carlos; F., Díaz, & H., Domínguez, [Show all 8 contributors for this article] (2025). Turning trash into treasure: Pressurized liquid extraction as a method for extracting bioactive compounds from spent mushroom substrate. Full text in Research Archive
• Soto, Luis Alejandro Romero; Klausen, Sarah Jasmin; Strætkvern, Knut Olav; Martín, Carlos; Shaojun, Xiong, & A., Liaqat, [Show all 7 contributors for this article] (2025). Deciphering biorefinery valorization routes for spent mushroom substrates: enzymatic saccharification and lignin recovery. Full text in Research Archive
• Martín, Carlos; J., Jönsson, Leif; P., Dixit,; F., Momayez,; Z., Dehghanmanshadi, & V., Benavente, [Show all 12 contributors for this article] (2025). Biorefining Norway spruce-based bioresources with the green solvent γ-valerolactone. Full text in Research Archive
• Dixit, Pooja; Benavente, V; Gustafsson, T.; Jönsson, Leif J. & Martín, Carlos (2024). γ-valerolactone (GVL) mediated production of biofuels and biochemical precursors from Norway spruce wood. Full text in Research Archive
• Martín, Carlos; Xiong, Shaojun; Passoth, Volkmar; Strætkvern, Knut Olav; Klausen, Sarah J. & Romero-Soto, Luis Alejandro (2024). Spent substrate from mushroom cultivation – potential feedstock for biorefinery exploitation. Full text in Research Archive
• Klausen, Sarah Jasmin; Diaz, F; Liaqat, Ayesha; Díaz-Reinoso, B; Torres, MD & Flórez-Fernández, N [Show all 8 contributors for this article] (2024). Recovery of antioxidant compounds from Lentinula edodes and Hericium erinaceus spent mushroom substrate by sequential extraction with microwave hydrodiffusion and gravity and supercritical carbon dioxide. Full text in Research Archive
• Romero-Soto, Luis Alejandro; Fernández, Lucía; Miranda, Diego A.; Strætkvern, Knut Olav & Martín, Carlos (2024). Exopolysaccharide production from underexplored agrowastes through microbial fermentation using bacterial isolates from a hypersaline environment. Full text in Research Archive
• Chen, Feng; Martín, Carlos & Xiong, Shaojun (2024). Preheating treatment of softwood to enable its use as substrate for an integrated production of mushroom and ethanol. Full text in Research Archive
• Miranda, Diego A.; Romero-Soto, Luis Alejandro; Jönsson, Leif J.; Carrasco, Cristhian & Martín, Carlos (2024). Exopolysaccharides from quinoa stalks: biosynthesis and in-depth characterization. Full text in Research Archive
• Akter, Afrina; Klausen, Sarah Jasmin; Romero-Soto, Luis Alejandro; Diaz, F; Dominguez, Herminia & Strætkvern, Knut Olav [Show all 7 contributors for this article] (2024). Pressurized liquid extraction of bioactive compounds from spent mushroom substrate of Lentinula edodes and Pleurotus ostreatus. Full text in Research Archive
• Yangin-Gomec, Cigdem; Horváth, Ilona Sárvári & Martín, Carlos (2023). Energy Production from Biomass Valorization. Energies. 16(11). doi: 10.3390/en16114300. Full text in Research Archive
• Martín, Carlos (2023). Spent substrate from mushroom cultivation – potential of an unexplored feedstock for biorefinery exploitation. Full text in Research Archive
• Xiong, Shaojun; Martín, Carlos & Chen, F (2023). Residual forest biomass for combined production of food and biofuel. Full text in Research Archive
• Klausen, Sarah J.; Falck-Ytter, Anne Bergljot; Strætkvern, Knut Olav & Martín, Carlos (2023). Extraction of bioactive compounds and saccharification of polysaccharides for valorization of spent mushroom substrate following a biorefinery approach. Full text in Research Archive
• Moreno-García, Jaime; Espinosa, Eduardo; Martín, Carlos; Moreno, Juan José & Ogawa, M (2023). Applying the biorefinery concept to “Tempranillo” grape stalks: analyzing by-product fractions and bioethanol production. Full text in Research Archive
• Miranda, Diego A.; Marin, Katherine; Sundman, Ola; Lundqvist, Jenny; Romero-Soto, Luis & Jönsson, Leif J. [Show all 8 contributors for this article] (2023). From lignocellulosic residues to biopolymers – poly(3-hydroxybutyrate) production from cellulosic hydrolysates of quinoa stalks using Halomonas boliviensis. Full text in Research Archive
• Miranda, Diego A.; Marin, Katherine; Sundman, Ola; Romero-Soto, Luis; Jönsson, Leif J. & Carrasco, Cristhian [Show all 7 contributors for this article] (2023). Biopolymers from crop residues – Poly(3-hydroxybutyrate) production from cellulosic hydrolysates of quinoa stalks using Halomonas boliviensis. Full text in Research Archive
• Gandla, Madhavi Latha; Tang, Chaojun; Martín, Carlos & Jönsson, Leif J. (2023). Enzymatic saccharification of lignocellulosic biomass. In Jeshke, Peter & Starikov, Evgeni B (Ed.), Agricultural Biocatalysis - Enzymes in Agriculture and Industry. Jenny Stanford Publishing. ISSN 9789814968478. p. 413–469. Full text in Research Archive
• Martín, Carlos; Rodriguez, Alejandro & Montagnaro, Fabio (2023). Introduction to the RSC Advances themed collection Chemistry in Biorefineries. 13(41), p. 28561–28563. doi: 10.1039/d3ra90087h. Full text in Research Archive
• Rodríguez, Alejandro; Espinosa, Eduardo & Martín, Carlos (2023). Special Issue “Lignocellulosic Biomass II”. Molecules. ISSN 1431-5157. 28(17). doi: 10.3390/molecules28176230. Full text in Research Archive
• Semaan, Georgeio; Kumar, Gopalakrishnan; Martín, Carlos & Jørgensen, KÃ¥re Bredeli (2026). Valorizing Mixed Lignocellulosic Waste: Integrated Process Design and Economic Assessment of Multi-Product Biorefineries. Universitetet i Stavanger. ISSN 9788284394497. Full text in Research Archive Show summary

  Fossil-based energy and chemical products have played a key role in driving economic prosperity for decades. The growing focus on sustainability, climate change, and environmental challenges has accelerated interest in transitioning to non-fossil-based and renewable fuels, chemicals, and energy. Non-edible lignocellulosic biomass is an abundant yet underutilized renewable resource with significant potential. However, lignocellulosic bioproducts often struggle to compete economically with petrochemical alternatives due to their high production costs, process inefficiencies, scale-up challenges, and limited price competitiveness. To avoid the environmental consequences of practices such as landfilling or open burning of lignocellulosic biomass waste, and to unlock its full economic value, systematic innovation in the process design and product portfolio of biorefineries is needed. While biorefineries hold strong commercial promise, profitability is essential. To attract and sustain investment, biorefineries must demonstrate financial viability, which is fundamental not only for their continued operation but also for their long-term survival. Biorefinery firms face constant pressure to deliver high-quality products and services at the lowest possible cost, as the risk of being outcompeted in a global market is ever-present. Although extensive research exists on the fractionation and valorization of lignocellulosic resources, few studies have focused on regional Norwegian biomass residues, particularly in multi-product biorefinery systems. Therefore, this gap warrants further investigation, beginning with labscale experimentation, followed by process simulation, and finally economic feasibility studies, to explore and validate the economic performance and operational viability of biorefineries at larger scales. In this work, a holistic, system-oriented approach was adopted to analyze integrated lignocellulosic biorefineries, with a particular focus on technical and economic aspects aimed at improving overall biorefinery performance. The study was structured in three parts. The first part involved laboratory-scale fractionation of a mixture of regional Norwegian lignocellulosic biomass, namely spent coffee grounds, tomato vine waste biomass from Lycopersicon esculentum, cucumber vine waste biomass from Cucumis sativus, and brewers’ spent grain, using a two-stage pretreatment consisting of dilute oxalic acid hydrolysis followed by organosolv delignification using ethanol as a solvent and oxalic acid as a catalyst. The objective was to evaluate the effectiveness of the two-stage pretreatment strategy using a mixed lignocellulosic feedstock by focusing on solid recovery, sugar solubilization, and delignification efficiency, as well as structural and compositional changes across pretreatment conditions. In the second part, insights from the experimental work were scaled up through process simulation and techno-economic modeling of a conceptual, multi-feedstock, multiproduct integrated biorefinery system. Thereafter, several production scenarios were assessed at varying capacities using both experimental data and literature-based assumptions to evaluate key financial indicators such as internal rate of return, net present value, and payback period, supported by sensitivity analyses. The third part examined Bitcoin mining as an ancillary income stream, assessing its potential to enhance competitiveness, diversify revenues, and strengthen the overall financial performance of biorefineries. To that end, four studies were conducted, each addressing a specific component of the viability and performance of integrated biorefineries using regional feedstocks. Paper I demonstrated that the two-stage pretreatment was broadly effective in fractionating the lignocellulosic biomass mixture. Dilute acid hydrolysis enabled substantial carbohydrate solubilization, achieving high sugar recoveries. However, some of the studied conditions were overly severe, leading to increased formation of sugar dehydration byproducts. In the second stage, ethanol organosolv delignification achieved moderate lignin removal while preserving cellulose integrity. Nonetheless, elevated pretreatment severities in both stages promoted sugar degradation and lignin condensation, resulting in diminished lignin recoveries. These findings underscore the importance of condition optimization to balance sugar recovery, delignification degree, and inhibitor formation, and suggest that while biomass blending holds potential, further refinement is needed to fully realize its benefits across sequential processing steps. Paper II scaled up experimental results from Paper I into a conceptual design for a regional multi-feedstock biorefinery, using SuperPro Designer® for process simulation, accompanied by a detailed techno-economic assessment. Six scenarios, varying by production scale and inclusion or exclusion of key process sections (namely, organosolv delignification and lactic acid fermentation), were evaluated. Only the large-scale (250 kt/year) designs achieved positive net present values and internal rates of return above standard investment thresholds, particularly when lactic acid was included. The organosolv stage significantly increased capital and solvent-related costs, thereby reducing overall profitability. Sensitivity and Monte Carlo analyses identified market prices for lactic acid and furfural as the most influential variables, followed by feedstock and solvent costs. The study concludes that commercial viability depends critically on production scale, integration of high-value coproducts, and efficient process configuration in lignocellulosic biorefineries. Paper III examined the conceptual feasibility of integrating Bitcoin mining as an auxiliary income stream with the potential to subsidize and incentivize biorefinery operations by generating additional revenue. This concept was built on the premise that internal energy flows within the biorefinery could be strategically redirected for alternative financial purposes. In this context, mining was positioned as an application for residual or externally sourced energy, enabling additional monetization alongside the continued production of traditional bio-based outputs. Estimates comparing the energy content of various biofuels in conjunction with different mining hardware efficiencies highlighted substantial variability in revenue potential. While integration may offer a pathway to enhanced biorefinery financial resilience, its viability remains highly dependent on site-specific conditions and calls for further assessment. Paper IV built on the perspective of Paper III by constructing a stochastic financial forecasting model to evaluate the associated costs and potential of integrating Bitcoin mining into biorefineries. Using sensitivity analysis and Monte Carlo simulations, the study quantified how profitability was affected by dynamic variables including electricity costs, mining hardware depreciation, Bitcoin price volatility, network hashrate, operational timing, and biorefinery-specific expenditures. Although the model demonstrated that profits from Bitcoin mining could, under certain optimal conditions, subsidize internal operational costs, this also introduced substantial financial risk, with the majority of simulated scenarios resulting in negative returns. The analysis underscored that even minor deviations in input assumptions, particularly around timing and cost estimation, could dramatically shift outcomes. No definitive conclusion was drawn regarding the overall viability of integration, as results were extremely sensitive to forecasting uncertainty and context-specific conditions. Finally, instead of relying solely on forward projections, profitability was estimated and backtested using historical data to understand what would have realistically happened if Bitcoin mining had been incorporated into the studied biorefinery system between January 1, 2021, to December 31, 2024. In aggregate, the four studies suggest that integrated biorefineries utilizing regional lignocellulosic waste biomass are technically feasible and may achieve economic viability, especially when co-production is strategically balanced across higher-value products. However, more empirical validation is needed to examine their long-term operational stability and cost dynamics before committing to a large-scale deployment. The integration of auxiliary income streams, such as Bitcoin mining, can generate additional revenues but carries significant uncertainty, volatility, and site-specific risk. The role of cross-sector integration via Bitcoin mining in enhancing economic competitiveness also warrants continued investigation.

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